Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells

Definitions

• the present invention relates to methods for characterizing cells and in particular to methods for determining whether a human cell is a hemopoietic cell and whether a human tissue cell is neoplastic.
• oncogenes in the development of human cancer has been amply demonstrated in recent years by the ability of these genes to cause tumorigenic conversion of rodent cells.
• other human genes which are not classified as oncogenes per se but which play important roles in the development and progression of cancer.
• One category of these genes encodes abundant structural proteins such as the actins and tropomyosins.
• the involvement of these abundant proteins in the neoplastic transformation process is suggested by the well documented observations that isoforms within these structural protein families are consistently modulated in transformation of avian, rodent and human cells.
• This second category of cancer-related genes is set apart from the so-called "oncogenes" because modulation of these genes in a transformation-sensitive manner is likely to result from regulatory processes activating transcription or translation rather than by direct activation through mutational processes.
• plastin An abundant phosphorylated polypeptide, plastin, is frequently expressed in human cancer cells of solid tissue but is not expressed in normal human fibroblasts. The same protein was one of the most abundant constitutively expressed proteins of human white blood cells. Thus, plastin may be associated with transformation of non-hemopoietic cells. Despite the reproducible identification of plastin and studies of its expression and polymorphic character, nothing was known of its molecular identity.
• NIMH4 or NC4
• l -plastin of fibroblasts were determined to be the same polypeptide (Leavitt et al . (1982), supra ).
• a method and reagents are provided for deter­mining whether a human cell is a hemopoietic cell and whether a human tissue cell is in a neoplastic state.
• Human cells which express only leukocyte-plastin ( l -plastin) are hemopoietic cells and human cells which express both l -plastin and tissue-plastin ( t -plastin) are neoplastic.
• the method can be performed using isoform-specific plastin nucleotide probes or isoform-­specific anti-plastin antibodies.
• a method for distinguishing human hemopoietic cells, normal human tissue cells and neoplastic human tissue cells is provided. The method is based on the observation that there are two isoforms of plastin, leukocyte-plastin ( l -plastin) and tissue-plastin ( t -plastin). Tissue cells express t -plastin, while neoplastic tissue cells additionally express l -plastin. Hemopoietic cells express l -plastin, but neither neoplastic nor normal hemopoietic cells express t -plastin.
• a cell that expresses t -plastin is a tissue cell; a cell that expresses only l -plastin is a hemopoietic cell; and a cell that expresses both t -plastin and l -plastin is a neoplastic tissue cell.
• Isoform-specific nucleic acid probes and isoform-­specific anti-plastin antibodies are also provided.
• the two isoforms of plastin, t -plastin and l -plastin have approximately 80% of their amino acids in common and the genes encoding the isoforms have about 60% of their nucleotides in common.
• the l -plastin isoform is polymorphic.
• t -plastin may actually be different isoforms of the protein, they will be referred to as the t -plastin isoform for purposes of this application, since each is distinguishable from the l -plastin isoform using the same nucleotide probe or antibody composition.
• a DNA fragment of at least about 1700 bp and fewer than about 50 kbp, usually fewer than 30 kbp, comprising a DNA sequence encoding a human plastin isoform or the 5′ and 3′ flanking non-coding regions is provided.
• the fragment may be a cDNA sequence comprising the doding and adjacent transcribed regions, usually of not more than about 5 kbp, or a genomic sequence including non-transribed regions of the gene.
• the isolation of cDNA sequences and genomic DNA sequences encoding l -plastin and t -plastin is described in detail in the Experimental section.
• Table 2 in the Experimental section provides the complete nucleotide sequences and the deduced amino acid sequences of cDNA fragments encoding t -plastin and l -plastin isolated from a ⁇ gt10 cDNA library of transformed human fibroblasts.
• the fragment finds use as a probe for detecting other human plastin-encoding genes or closely-related genes in human or other species and for detecting mRNA expressing human plastin.
• the fragment may also be used to express the encoded plastin isoform by preparing by conventional means an expression construct containing the fragment under the transcrip­tional and translational control of a promoter.
• the promoter may be a eukaryotic promoter for expression in a mammalian cell. In cases where one wishes to expand the promoter or produce the peptide isoforms or fragments thereof in a prokaryotic host, the promoter may also be a prokaryotic promoter. Usually a strong promoter will be employed for high level transcription and expression.
• the expression construct may be part of a vector capable of stable extrachromosomal maintenance in an appropriate cellular host or may be integrated into host genomes. Normally, markers are provided with the expression construct which allow for selection of a host containing the construct.
• the marker may be on the same or a different DNA molecule, desirably, the same DNA molecule.
• the isoform may serve as a marker or markers such as resistance to a cytotoxic agent, complementation of an auxotrophic host to prototrophy, production of a detectable product etc. will be convenient.
• the expression construct can be joined to a replication system recognized by the intended host cell.
• Various replication systems include viral replication systems such as retroviruses, simian virus, bovine papilloma virus, or the like.
• the constuct may be joined to an amplifiable gene, e.g. DHFR gene, so that multiple copies of the fragment may be made.
• Introduction of the construct into the host will vary depending on the construct and can be acheived by any convenient means.
• a wide variety of hosts may be employed for expression of the peptides, both prokaryotic and eukaryotic.
• Nucleotide sequences of at least about 20 nucleotides and not more than about 50 kbp, usually less than 30 kbp, which include at least about 20 consecutive nucleotides complementary to a DNA sequence encoding a plastin isoform or the adjacent non-coding regions also find use as probes.
• the nucleotides will be complementary to the transcribed portions of the plastin gene, desirably the coding region, however, the untranscribed regions find use in isolating genomic sequences.
• the probe will have at least about 50, more usually about 100, generally 500 nt complementary nucleotides.
• the probe when using probes of less than about 500 nt, will share not more than about 60% homology with a DNA sequence encoding the other plastin isoform, usually not more than about 50% homology.
• the probe will be complementary to at least a portion of a DNA sequence encoding the N-terminal 20 amino acids, usually the N-terminal 15 amino acids of the plastin isoform.
• the probe will include a sequence encoding the N-terminal 20 amino acids of the plastin isoform.
• the probe will include an oligonucleotde sequence in Table 1B.
• the probes may be used to detect genes encoding plastin or to express at least a portion of the plastin isoform. Usually, however, the probe will be a DNA sequence which finds use to determine whether a cell produces mRNA expressing the isoform.
• a substantially pure composition of a plastin isoform may be produced by expressing a DNA fragment of this invention.
• Peptides corresponding to a portion of the plastin isoform peptide sequence can be produced by recombinant technology or can be chemically synthesized.
• a peptide of less than about 100 amino acids comprises at least about 10 consecutive amino acids, more usually at least 15 consecutive amino acids, from the amino acid sequence of a plastin isoform or sequences immunologically cross-reactive therewith.
• the peptide will have at least about 5 consecutive amino acids, usually 10, more usually 15, of the N-terminal about 15 to about 20 amino acids of the plastin isoform sequence.
• Peptides of less than about 50 amino acids, more usually less than about 30 amino acids, and comprising about 15 to about 20 amino acids of the N-terminal sequence of the plastin isoform may find use to induce isoform-specific anti-plastin antibodies.
• the peptide sequence will include at least about 10 consecutive amino acids form the sequence MLDGDRNKDGKISFDEFVYI or MATGDLDQDGRISFDEFIKI.
• the peptides may also be used to quantify a peptide isoform as controls or as analyte analogue in competitive inhibition analyses, to determine the specificity of an antibody composition or to purify an antibody composition.
• peptides used for production of isoform-specific antibodies will share not more than about 60% homology, usually not more than about 50% homology, desirably 40% homology or less, with the other plastin isoform.
• the first 15 to 20 amino acids of the N-terminus of plastin isoforms share fewer common amino acids than many other portions the isoform sequence of the same length.
• the N-terminal sequence of the isoform is conveniently included in the peptides.
• An isoform-specific anti-plastin antibody composition reacts with a plastin isoform and exhibits substantially no reaction with the other plastin isoform.
• the antibody composition desirably has an affinity for the isoform suitable for detection of the isoform on a solid substrate, particularly a Western blot, and exhibits only a backgound level of binding with the other isoform.
• the antibody affinity required for detection of a plastin isoform using a particular immunoassay method will not differ from that required to detect other polypeptide analytes.
• the antibody composition may be polyclonal or monoclonal, desirably monoclonal.
• Isoform-specific antibodies can be produced by a number of methods.
• Polyclonal antibodies may be induced by administering an immunogenic composition comprising a peptide of this invention to a host animal. Preparation of immunogenic compositions may vary depending on the host animal and are well known.
• the peptide may be conjugated to an immunogenic substance such as KLH or BSA or provided in an adjuvant or the like.
• the induced antibodies can be tested to determine whether the composition is isotype-­specific. If a polyclonal antibody composition does not provide the desired specificity, the antibodies can be purified to provide an isoform-specific composition by a variety of conventional methods.
• the composition when one isoform is used to induce antibodies, the composition can be contacted with the other isoform affixed to a solid substrate to remove those antibodies which bind to the other isoform. Either prior to or following that purification, if desired, the composi­tion can be purified to reduce binding to other substances by contacting the composition with the desired isoform affixed to a solid substrate. Those antibodies which bind to the desired isoform are retained. Purification techniques using peptides or antibodies affixed to a variety of solid substrates such as affinity chromatogaphy materials including Sephadex, Sepharose and the like are well known.
• Monoclonal isoform-specific anti-plastin antibodies may also be prepared by conventional methods.
• a mouse can be injected with an immunogenic composition comprising a peptide of this invention and spleen cells obtained. Those spleen cells can be fused with a fusion partner to prepare hybridomas.
• Anti­bodies secreted by the hybridomas can be screened to select a hybridoma wherein the antibodies react with one plastin isoform and exhibit substantially no reaction with the other plastin isoform.
• Hybridomas that produce antibodies of the desired specificity are cultured by standard techniques. Hybridoma preparation techniques and culture methods are well known and constitute no part of the present invention.
• a method of determining whether a cell is hemopoietic comprises determining whether l -plastin, but not t -plastin is present in the cell.
• Cells that have l -plastin but not t -plastin are hemopoietic.
• cells could be deter­mined to be hemopoietic by a variety of means.
• cells can be determined to be hemopoietic histologically or by detecting the presence of a variety of markers specific for different types of hemopoietic cells.
• the present technique is particu­ larly advantageous in that a marker common to a plurality of types of hemopoietic cells had not previously been found.
• l -plastin is a marker common to all cells of hemopoietic origin. Further, all cells that express l -plastin but fail to express t -plastin are hemopoietic cells.
• a method for determining whether a tissue cell is in a neoplastic state comprises determining whether l -plastin is present in the cell.
• Tissue cells which express l -plastin are neoplastic.
• the cell can be determined to be a non-hemopoietic cell by any of a number of methods, conveniently, the cell will be determined to be a tissue cell by detecting the presence of t -plastin.
• a cell which expresses both t - and l -plastin is a neoplastic tissue cell.
• the cell may be neoplastic. That is, a positive result (presence of both t - and l -plastin) indicates that a cell is neoplastic, but a negative result is not conclusive.
• an isoform-specific plastin probe or an isoform-specific anti-plastin antibody can be used to detect the presence of mRNA or plastin peptide isoform, respectively.
• the probe When using a probe, the probe will be hydridized to mRNA from the cell under stringent hybridization conditions.
• An exemplary method of detecting mRNA encoding a plastin isoform by a Northern blot analysis is described in the Experimental section.
• Detecting mRNA is conveniently performed on a cell which has been cultured to provide a number of identical cells sufficient for mRNA analysis.
• the amount of l -plastin mRNA present in a cell comprises about 0.1% to about 200% of the amount of t -plastin mRNA. Therefore, the sample being analyzed has been determined to contain only a small percentage of hemopoietic cells, desirably less than 0.001% by weight, the presence of l -plastin mRNA in a substantial percentage of the tissue cells, usually at least about 0.1%, desirably 1.0% or more, can be distinguished from l -plastin mRNA in the contaminating hemopoietic cells.
• the presence of t - or l -plastin can be detected using isoform-specific anti-plastin antibodies.
• isoform-specific anti-plastin antibodies A variety of methods of detecting polypep­tides with specific antibodies are well known. Since plastin comprises a major component of the cell cytosol, determination of which isoforms are present in particular cells is conveniently performed by preparing the cells for staining by standard histologic techniques such as embedding formalin-fixed cells in paraffin.
• the isoform specific antibodies can be conjugated to distinguishable labels such as rhodamine and fluorescein and used simultaneously to stain the cells. Such methods unambiguously indicate the presence of both isoforms within a cell.
• samples could be sequentially stained with each of the isoform-­specific antibodies.
• samples could be sequentially stained with each of the isoform-­specific antibodies.
• a statistically larger percentage of cells stain with anti- l -plastin than do not stain with anti- t -plastin there are cells in the sample that contain both l - and t -plastin. That or similar methods may also find use with samples substantially free from hemopoietic cells or samples having a significant percentage of neoplastic cells.
• a preferred assay is a Western blot which definitively demonstrates that the correct peptide has been detected through the combination of the use of gel electrophoresis and immunoassay analysis.
• other immunoassay methods may also find use. Numerous quantitative immunoassay methods for detecting a peptide in a bodily fluid are known. An assay method has the following elements.
• the method comprises combining the sample with the isoform-specific antibody and detecting the presence of isoform-specific antibody-peptide complex as indicative of the presence of the peptide. in the sample.
• the particular manner in which the peptide is detected is not significant for the purpose of this invention so long as the method provides the desired degree of sensitivity and reliability.
• the assay conditions and reagents may be any of a variety found in the prior art.
• the assay may be heterogeneous or homogeneous, usually heterogeneous, conveniently a sandwich assay.
• the assay will usually employ solid phase-­affixed isoform specific anti-plastin antibodies.
• the antibodies may be polyclonal or monoclonal, usually monoclonal.
• the solid phase-affixed antibodies are combined with the sample. Binding between the anti­bodies and sample can be determined in a number of ways. Complex formation can be determined by use of soluble antibodies specific for the isoform to be detected.
• the antibodies can be labeled directly or can be detected using labeled second antibodies specific for the species of the soluble antibodies.
• Various labels include radionuclides, enzymes, fluorescers or the like.
• the assay will be an enzyme-linked immunosorbant assay (ELISA) in which monoclonal antibodies specific for different epitopes of the plastin isoform are used as the solid phase-affixed and enzyme labeled, soluble antibodies.
• ELISA enzyme-linked immunosorbant assay
• the assay may be based on competitive inhibition, where plastin in the sample competes with a known amount of a plastin isoform for a predetermined amount of isoform-specific anti-plastin antibody.
• plastin in the sample competes with a known amount of a plastin isoform for a predetermined amount of isoform-specific anti-plastin antibody.
• any of the plastin isoform present in the sample can compete with a known amount of the labeled plastin isoform or isoform analogue for antibody binding sites.
• the amount of labeled isoform affixed to the solid phase or remaining in solution can be determined.
• proteins were electroblotted from analytical two-dimensional gels onto chemically-modified glass fiber filter paper and detected by fluorescent staining. Plastin spots were cut out and inserted into the sequenator cartridge for direct sequence analysis (Aebersold et al . (1986), supra ; Aebersold et al . (1987a), supra).
• the amino acid sequence analysis was fperformed on an automated Caltech gas-phase sequenator (Hewick et al ., J. Biol. Chem (1981) 256 :7990-7997).
• Proteins in a total cell lysate of CEM lymphoblastoid cells were separated by two-dimensional gel electrophoresis and electroblotted onto nitrocellu­lose. Plastin containing spots were excised and enzymatically cleaved on the nitrocellulose matrix (Aebersold et al . (1987b), supra ). The resulting peptides were separated by narrow-bore reversed-phase HPLC and individual peptide fragments were sequenced in a modified Caltech gas-phase sequenator (Kent et al ., Biotech. (1987) 5 :314-321). Four peptide sequences that were unambiguously identified are listed in Table 1. TABLE 1 A.
• a degenerate oligonucleotide 20-mer corresponding to one of the four oligopeptides was synthesized and used to screen a ⁇ gt10 cDNA library of transformed (HuT-14) human fibroblasts (Lin et al ., J. Mol. Cell. Biol. (1988) 8 :160-168). From approximately 10,000 recombinants, the probe selected a single clone, P4.
• RNAs were prepared by the guanidine hydrochloride method (as previously described in Gunning et al ., J. Mol. Evol. (1984) 20 :202-214). Five microgram of each RNA was electrophoresed in a 1% agarose gel containing 50 mM morpholinepropanesulfonic acid (pH 7), 1 mM EDTA, and 2.2 M formaldehyde; blotted onto nitrocellulose; and hybridized with nick-­translated probes (Rigby et al ., J. Mol. Biol. (1977) 113 :237-251).
• Hybridization proceeded overnight at 65°C in 4x SSC (1x SSC is 150 mM NaCl and 15 mM sodium citrate, pH 7), 5x Denhardt's solution (Denhardt, Biochem. Biophys. Res. Comm. (1966) 23 :641-646), 50 mM phosphate buffer (pH 7), 10% (w/v) dextran sulfate, and 2 X 106 cpm/ml probe. Washing was carried out twice in 1x SSC and 0.1% (w/v) sodium dodecyl sulfate (SDS) at room temperature for 5 min each, and twice in 0.5x SSC and 0.1% SDS at 65°C for 30 min each.
• 4x SSC is 150 mM NaCl and 15 mM sodium citrate, pH 7
• 5x Denhardt's solution Denhardt, Biochem. Biophys. Res. Comm. (1966) 23 :641-646
• the cDNA insert of P4 was 4.3 kilobases (kb) in length. (This large cDNA was later determined to be comprised of two unrelated cDNA fragments of 1.0 kb and 3.3 kb that were ligated during the construction of the library.) A 2-kb Hin dIII fragment within the 3.3-kb cDNA insert of P4 hybridized to an mRNA of 3.4 kilobases that was more abundant in HuT-14 than in normal KD fibroblasts, and not detectable in CEM T-lymphocytes.
• cDNA was cloned into M13mp9 (Messing, J. Methods in Enzymol. (1983) 101 :20-78). Progressive deletion clones were prepared by the method described by Dale et al ., Plasmid (1985) 13 :31-40. Sequencing was done by the method described by Sanger et al ., Proc. Natl. Acad. Sci. USA (1977) 74 :5463-5467. Table 2 illustrates the coding sequences and deduced amino acid sequences of P4 cDNA (top, t -plastin) and P107 cDNA (bottom, l -plastin. Identical nucleotides between the two sequences are indicated by double dots. As shown in the table, the cDNA sequences share about 60% homology, while the peptide sequences share about 80% homology.
• Total cellular RNA was prepared by the guanidine hydrochloride method, as described previously (Gunning et al ., supra ).
• PolyA+ RNA was prepared by oligo(dT)-cellulose chromatography (Aviv et al ., Proc. Natl. Acad. Sci. USA (1972) 69 :1408-1412).
• the purified cDNA fragment was bound to nitrocellulose paper essentially as described by Parnes et al ., Proc. Natl. Acad. Sci. USA (1981) 78 :2253-2257, except that the DNA solution was not subjected to boiling and was added to nitrocellulose paper with the aid of a Minifold (Schleicher & Schuell, Inc., Kenne, NH).
• Hybridization to mRNA and elution of hybridized mRNA were done as described by Maniatis et al ., Molecular cloning, a laboratory manual , Cold Spring Harbor Lab., Cold Spring Harbor, NY (1982), except that tRNA was not included in the hybridization solution or during the elution of mRNA.
• the eluted mRNA was precipitated in the presence of calf liver tRNA (15 Mg/ml) in 70% ethanol.
• the mRNA was suspended in water and subjected to in vitro translation. All in vitro translation experiments were carried out in a rabbit reticulocyte system purchased from New England Nuclear Corp. (Boston, MA).
• the translated products were then electrophoresed in a two-dimensional polyacrylamide gel and visualized by autoradiography as described previously (Lin et al ., supra ).
• the method of 2-D gel electrophoresis and translation of mRNAs selected by hybridization to cDNAs has been described previously (Lin et al ., supra ).
• the samples in each gel were as follows.
• Polypeptide X identified as a 70 kd heat shock response polypeptide in fibroblasts and leukocytes, served as a 2-D gel marker for the more acidic isoform of t -plastin in Gels C and E since it has the same pI and exhibits a slightly higher M r .
• the most prominent translation products were two proteins ( t -plastins) migrating to more basic isoelectric points than the polypeptide recognized as plastin in the 2-D gel (Gels F, G). These two proteins were abundantly synthesized in both normal and transformed human fibroblasts (Gels B, C), but were undetectable in white blood cells (Gels A and E). As these two proteins were apparently similar to plastin, the 2-kb Hin dIII fragment of P4 cDNA was used as a probe to rescreen the HuT-14 cDNA library to find related cDNAs.
• This in vitro synthesized polypeptide was determined to be electrophoretically identical to l -plastin by mixing the in vitro translation sample in Gel H with labeled unfractionated HuT-12 cellular proteins (Leavitt et al . (1986), supra ) shown in Gel J.
• This in vitro translated l -plastin which was in excess of the endogenous HuT-12 l -plastin, was superimposed upon the endogenous l -plastin following 2-D gel electrophoresis (Gel K). It was therefore concluded that P107 is the true cDNA clone of l -plastin, whereas P4 cDNA encoded a separate polypeptide isoform, t -plastin, closely related to l -plastin.
• P107 cDNA DNA sequencing of P107 cDNA revealed a coding sequence closely related to that of the initially characterized P4 clone (Table 2).
• This P107 cDNA sequence contained the amino acid residues determined by protein sequence analysis of plastin isolated from CEM lymphocytes in the positions where discrepancies had existed between protein sequence and P4 cDNA sequence.
• Each coding sequence encoded a polypeptide of 570 amino acids with molecular weight of 64 kilodaltons. This value (64kd) is slightly different from the observed M r value of l -plastin and the two t -plastin polypeptides (M r 68,000) in a 2-D gel.
• Post-transitional modification of the polypep­tides, such as glycosylation, may account for this discrepancy.
• a panel of cultured human cell strains was surveyed with Northern analysis for l -plastin (Blot A) and t -plastin mRNA (Blot B). The methods used were as described for Table 1. Two identical Northern blots, A and B, were hybridized with P32-labeled cDNAs of P107, and P4, respectively, to analyze plastin expression. Each blot contained cellular RNAs of the following cell strains.
• l -plastin mRNA and protein were not detected in the diploid, non-neoplastic human fibroblast strains KD and R17 but both strains exhibited t -plastin mRNA and protein (Gel B). Greater than 50 additional diploid human fibroblast strains derived from embryonic lung, skin, foreskin, and gingiva from normal individuals and from patients with various genetic diseases such as Huntington's disease, Bloom's syndrome, Ataxia telangiectasia, and retinoblastoma were examined. Without exception all diploid fibroblast strains exhibited expression of t -plastin in 2-D protein profiles, but not l -plastin.
• HuT-14T, an even more tumorigenic substrain of HuT-14 (Leavitt et al ., Cell (1982) 28 :259-2268, Leavitt et al ., J. Mol. Biol. (1986) 6 :2721-2726) was not elevated further in l -plastin expression.
• a second osteogenic sarcoma, MG63, and a retinoblastoma tumor cell line GM 1231A did not exhibit either the l -plastin mRNA transcript or the l -plastin polypeptide. All of the cell strains listed above expressed relatively constant levels of t -plastin mRNA transcripts and polypeptides except for the retinoblastoma cell line which exhibited no detectable plastin mRNA or protein at all.
• l -Plastin expression in additional neoplastic human cell strains derived from ovarian carcinoma, endometrial carcinoma, choriocarcinoma tumors and in vitro transformed keratinccytes was observed.
• some established cell lines derived from an ovarian carcinoma, an adenocarcinoma of the cervix (Hela), a Wilm's tumor and a colon carcinoma do not appear to express l -plastin.
• l -plastin In all 17 independent transformed (neoplastic) human cell lines derived from connective, epithelial and endothelial tissues, expressed l -plastin while 12 cell lines derived from other tumor sources did not exhibit l -plastin expression.
• retinoblastoma tumors which have deleted the l -plastin linked ret locus are included in these 12 l -plastin-negative cell lines.
• One of the retinoblastoma tumor cell lines, GM1231A is distinguished from all the other cell types in that it did not express either l - or t -plastin.
• the three lymphocyte cell lines CEM and Molt-4 both T-cell leukemias
• AG1484 a transformed B-lymphoblast cell line derived from the same patient as the retinoblastoma GM1231A, synthesized high levels of l -plastin mRNA, but no detectable t -plastin mRNA.
• Leukemic cell lines such as CEM and normal leukocytes such as peripheral blood leukocytes (Goldstein et al . (1985b), supra ) and cultured monocytes synthesize the highest levels of l -plastin protein (Goldstein et al . (1985b), supra ), but no t -plastin protein.
• lymphoblastoid cell lines More than 20 different lymphoblastoid cell lines were examined, the promyelocytic cell line HL-60 (Anderson et al ., Cancer Res. (1985) 45 :4995), red blood cells, peripheral blood leukocytes (PBLs) from more than 20 individuals, and subfractions of PBLs including T-cells, NK cells, granulocytes, polymorphonuclear leukocytes, and monocytes (Goldstein et al . (1985b), supra ). Without exception, all cells of hemopoietic origin except fully differentiated red blood cells exhibited abundant synthesis of l -plastin (Goldstein et al . (1985b), supra ), and failed to express t -plastin.
• Genomic DNA was prepared by the method described by Maniatis et al ., Cell (1978) 15 :687-701. Ten microgram of each genomic DNA was digested with Hind III to completion, electrophoresed in a 0.7% agarose, and blotted onto nitrocellulose.
• An eight-­fold degenerate 20 nucleotide probe described in Table 1B (Oligo Probe - Antisense) was designed from an l -plastin peptide sequence that exhibited a relatively low degeneracy. This oligonucleotide was synthesized using an ABI solid phase synthesizer. To further minimize degeneracy, preferred codon usage for valine and the weak base pair stabilizing characteristics of inosine (I) in the isoleucine codon (Ohtsuka et al ., J. Biol. Chem. (1985) 260 :2605-2608) were used.
• the oligonucleotide probe was end labeled with 32P and used to screen the HuT-14 cDNA library as previously described (Lin et al ., supra ) except that hybridization was performed at 42°C and the hybridized filters were washed at room-temperature with 2x SSC.
• Hybridization and washing conditions were identical to those described for Table 1.
• Two identical genomic blots were hybridized with 32P-labeled cDNAs of P4 and P107, respectively.
• Each blot contained Hind III-digested genomic DNAs of KD (Lane 1), HuT-12 (Lane 2), HuT-14 (Lane 3), peripheral blood lymphocytes (Lane 4), B lymphoblast strain AG1484 (Lane 5), and retinoblastoma tumor cell strain GM1231A (Lane 6). Size markers were Hind III fragments of phage DNA. All human cell strains are described above.
• peripheral blood leukocytes derived from a healthy human donor had two Hin dIII fragments of 6.5 kb and 6.0 kb instead of the single band of 6.5 kb found in the other five genomic DNA samples.
• This individual's peripheral blood leukocytes synthesized the variant (polymorphic) form of the l -plastin (Goldstein et al . (1985b, supra ) in addition to the normal charge species of l -plastin.
• the 6.0 kb fragment therefore may be indicative of this variant form of l -plastin.
• the genomic DNA of the B lymphoblastoid cell line derived from the retinoblastoma patient exhibited approximately a 50% reduction in hybridization of l -plastin restriction fragments compared to the other five genomic DNA samples including the retinoblastoma tumor from the same patient.
• the reduced copy number of l -plastin genomic fragments apparent for this cell line was consistent with the observation that the l -plastin gene is linked to the retinoblastoma locus because one of the retinoblastoma alleles has been lost as a result of a large deletion around the retinoblastoma locus on chromosome 13.
• l -plastin genomic sequences did not appear to be reduced to the same extent in the genome of the retinoblastoma of the same patient, ruling out the possibility that the l -plastin gene was tightly linked to the retinoblastoma locus.
• the plastin gene family that is described above encodes a truly novel set of at least two related but distinct proteins whose expression distinguished cells of solid tissue from hemopoietic or leukocyte cells.
• l -Plastin is a stable protein that is synthesized constitutively at a very high rate in a majority of subtypes of peripheral blood leukocytes and is one of the 10 or 20 most abundant proteins of these normal leukocyte cells.
• t -Plastin currently defines two equally abundant polypeptide species in fibroblasts, epithelial and endothelial cells that have the same molecular weight as l -plastin, but are slightly more basic.
• the present invention provides novel methods of identifying hemopoietic cells of all types.
• the invention also provides methods and reagents for determining tissue cells which are in a neoplastic state.
• the presence of the plastin isoform indicative of the particular states is readily identified using either isoform-specific nucleotide probes or isoform-­specific anti-plastin antibodies.

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• 1989
  • 1989-06-06 EP EP19890110218 patent/EP0345726A3/fr not_active Withdrawn
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